Recent evidence suggests that the cellular senescence machinery and the decline of the regenerative capacity of certain self-renewing stem cell compartments may contribute importantly to mammalian aging. Although derepression of INK4A/ ARF locus is thought to be an intrinsically linked event, very little is known about the specific mechanisms responsible for modulation of p16INK4 and p19ARF expression. Preliminary work from the PI's laboratory suggests that Slug, a zinc-finger transcription factor known to affect cell survival and the epithelial-mesenchymal transition in a variety of tissues, directly inhibits p16INK4 transcription, to the extent that Slug deficiency leads to cellular senescence and premature aging in vitro and in animal models. We therefore hypothesize that Slug influences aging by restraining cellular senescence and sustaining the regenerative potential of adult stem and progenitor cells, primarily through direct negative regulation of the p16INK4 gene. These developments promote our long-term objective -- to discover novel molecular pathways underlying the senescence of both differentiated and stem and progenitor cells -- and have led to three immediate research aims to determine the precise contribution of Slug to organismal aging. Aim 1 will establish the extent to which p16INK4a contributes to accelerated senescence in Slug-deficient MEFs using both knockdown and knockout technologies. Aim 2 will attempt to elucidate the molecular mechanisms by which Slug negatively regulates expression of p16INK4a, by relying on chromatin immunoprecipitation and electrophoretic mobility shift assays. Aim 3 will define the functional role of Slug in tissue senescence and the regenerative potential of adult stem and progenitor cells. These experiments will use a series of genetically modified animal models as well as hematopoietic and skeletal muscle stem cells from Slug and p16INK4a- deficient mice. This proposal is innovative because it exploits a transcription factor, Slug, with unique regulatory activity at the INK4a/ ARF locus. The likelihood that a better understanding of the in vivo regulation of this locus, and hence of mammalian aging, will emerge from our project appears high.